Sexual dimorphism in the control of amebic liver abscess in a mouse model of disease

Abstract

Amebic liver abscess (ALA) is the most common extraintestinal manifestation of human infection by the enteric protozoan parasite Entamoeba histolytica. In contrast to intestinal infection, ALA greatly predominates in males but is rare in females. Since humans are the only relevant host for E. histolytica, experimental studies concerning this sexual dimorphism have been hampered by the lack of a suitable animal model. By serial liver passage of cultured E. histolytica trophozoites in gerbils and mice, we generated amebae which reproducibly induce ALA in C57BL/6 mice. Interestingly, all animals developed ALA, but the time courses of abscess formation differed significantly between the genders. Female mice were able to clear the infection within 3 days, whereas in male mice the parasite could be recovered for at least 14 days. Accordingly, male mice showed a prolonged time of recovery from ALA. Immunohistology of abscesses revealed that polymorphonuclear leukocytes and macrophages were the dominant infiltrates, but in addition, gamma,delta-T cells, NK cells, and natural killer T (NKT) cells were also present at early times during abscess development, whereas conventional alpha,beta-T cells appeared later, when female mice had already cleared the parasite. Interestingly, male and female mice differed in early cytokine production in response to ameba infection. Enzyme-linked immunospot assays performed with spleen cells of infected animals revealed significantly higher numbers of interleukin-4-producing cells in male mice but significantly higher numbers of gamma interferon (IFN-gamma)-producing cells in female mice. Early IFN-gamma production and the presence of functional NKT cells were found to be important for the control of hepatic amebiasis as application of an IFN-gamma-neutralizing monoclonal antibody or the use of NKT knockout mice (Valpha14iNKT, Jalpha 18(-/-)) dramatically increased the size of ALA in female mice. In addition, E. histolytica trophozoites could be reisolated from liver abscesses of Jalpha18(-/-) mice on day 7 postinfection, when wild-type mice had already cleared the parasite. These data suggest that the sexual dimorphism in the control of ALA is due to gender-specific differences in early cytokine production mediated at least in part by NKT cells in response to E. histolytica infection of the liver.

FIG. 1.

Comparison of amebic liver abscess progression in male and female C57BL/6 mice. (A) Eight animals of each gender were intrahepatically challenged with 10⁵ E. histolytica trophozoites at each time. Animals were sacrificed at the times indicated, and the relative sizes of the abscesses were calculated by using the following scores: 0, no abscess; 1, pinhead; 2, <5.0 mm; 3, >5.0 mm. According to the Mann-Whitney U test, female mice had significantly smaller abscesses than male mice on day 7 (P < 0.05), on day 14 (P < 0.0001), and on day 21 (P < 0.0001). (B) Reisolation of E. histolytica trophozoites from liver abscess lesions of C57BL/6 mice. Infected animals were sacrificed at the times indicated, the entire liver was removed from each animal, and abscesses were dissected and dissolved in TYI-S-33 medium. The bars indicate the percentages of animals with positive ameba cultures. According to a paired t test, there were significant differences between male and female mice on day 7 (P < 0.0001) and on day 14 (P < 0.005). *, P < 0.05; **, P < 0.005; ***, P < 0.0001.

FIG. 2.

Immunostaining of immune cells in abscesses of mice with ALA from day 3 postinfection (A to C) and from day 7 postinfection (D and E). (A) γ,δ-T cells (anti-γ,δ-TCR) (magnification, ×63); (B) NK cells (anti-DX5) (magnification, ×63); (C) NKT cells (anti-NK-1.1 cells that coexpress CD3) (magnification, ×63); (D) polymorphonuclear leukocytes (anti-GR1) (magnification, ×20) that were found in areas of dense cell infiltration in the demarcation wall and around the central abscess region; (E) monocytes (anti-CD11b) (magnification, ×20) that are the main cells found in the abscess center; (F) conventional α,β-T cells (anti-α,β-TCR) (magnification, ×20) that are detectable in the abscess center, as well as in distinct areas at the abscess-demarcating margin.

FIG. 3.

Time courses of IL-4 (A) and IFN-γ (B) production in male and female mice with ALA. Cytokine-producing spleen cells were determined by ELISPOT assays. Spleen cells from male or female mice with ALA were cultivated on antibody-coated plates for 24 h either in the presence of medium alone or in the presence of anti-CD3. Plates were developed with the appropriate secondary antibodies, followed by the detection reagent. The number of cytokine-producing cells is expressed in spot-forming units (SFU).

FIG. 4.

Amebic liver abscesses in female mice after immunodepletion of IFN-γ. Ten female mice per group were immunized by intraperitoneal application of either 150 μl of PBS, 500 μg of neutralizing monoclonal antibody against IFN-γ (anti-IFN-γ), or 500 μg of isotype-matched control antibody (IgG isotype control). One day later, animals were challenged by intrahepatic inoculation of virulent E. histolytica trophozoites. The sizes of abscesses were scored on day 7 postinfection.

FIG. 5.

IFN-γ production in female Vα14iNKT cell-deficient Jα18^−/− mice after induction of ALA. Cytokine production from spleen cells was determined by ELISPOT assays. Spleen cells either from uninfected C57BL/6 and Vα14iNKT cell-deficient Jα18^−/− mice or from the same mouse strains 7 days after intrahepatic challenge with E. histolytica trophozoites (right panel) were cultivated on antibody-coated plates for 24 h in the presence of medium alone (−) or in the presence of anti-CD3 (+). Plates were developed with the appropriate secondary antibody, followed by the detection reagent. The number of cytokine-producing cells is expressed in spot-forming units (SFU). Each group consisted of five animals. p.i., postinfection.